WO2017096221A1 - Anticorps largement neutralisants anti-vih bispécifiques - Google Patents

Anticorps largement neutralisants anti-vih bispécifiques Download PDF

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WO2017096221A1
WO2017096221A1 PCT/US2016/064713 US2016064713W WO2017096221A1 WO 2017096221 A1 WO2017096221 A1 WO 2017096221A1 US 2016064713 W US2016064713 W US 2016064713W WO 2017096221 A1 WO2017096221 A1 WO 2017096221A1
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antigen
binding site
seq
antibody
3bnc117
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PCT/US2016/064713
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Jeffrey V. Ravetch
Stelios BOURNAZOUS
Michel Nussenzweig
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The Rockefeller University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This invention generally relates to bispecific antibodies and, in particular, relates to bispecific anti-HIV broadly neutralizing antibodies and uses thereof.
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • HIV-1 is the most common and pathogenic strain of the virus.
  • the development and use of potent neutralizing antibodies against HIV-1 has been a challenge for efforts to prevent or control HIV-1 infection.
  • HIV-1 presents several unique structural and functional determinants that conventional antibody strategies must overcome to block viral entry to target cells.
  • These immune evasion mechanisms greatly compromise the host's capacity to mount broadly neutralizing potent antibody responses.
  • early clinical studies have identified a small fraction of infected individuals that develop affinity matured antibodies with broad activity against diverse, cross-clade virus isolates (Simek, M.D. et al. J Virol 83, 7337-7348 (2009)).
  • mAbs monoclonal antibodies
  • env HIV-1 envelope glycoprotein
  • the relatively high virus mutation rate, its capacity to remain latent for several years even in chronically treated patients, as well as the chronicity of infection greatly favor the development of bNAb-resistant virus mutants resulting from selection pressure by the administered antibody.
  • the envelope glycoprotein of HIV-1 (env) is present at remarkably low density on the virus surface, thereby precluding high avidity concurrent interactions of both IgG Fab arms (Klein, J.S. & Bjorkman, P.J. PLoS Pathog 6, el000908 (2010)).
  • This characteristic is unique to HTV- 1 and is a key immune evasion mechanism that greatly reduces the antiviral activity of passively administered bNAbs.
  • the invention addresses the aforementioned unmet need by providing novel bispecific antibodies.
  • the invention provides a bispecific antibody comprising (i) a first antigen-binding site that binds to a first epitope, said first antigen-binding site comprising a first heavy chain and a first light chain; (ii) a second antigen-binding site that binds to a second epitope, said second antigen-binding site comprising a second heavy chain and a second light chain.
  • Either or both of the first heavy chain and the second heavy chain can comprise a variant hinge region that (a) is derived from a wild type IgG3 hinge region and (b) has fewer CH-1 proximal cysteine residues than the wild type IgG3 hinge region as compared to a reference antibody containing the wild type IgG3 hinge region.
  • the wild type IgG3 hinge region can comprise the sequence of SEQ ID No. 2.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) or the majority (i.e., more than 50%) of the CH-1 proximal cysteine residues (i.e., cysteine residues proximal or closers to the variable regions) of the wild type IgG3 hinge region are substituted to another residue (i.e., any non-cysteine residue including but not limited to serine).
  • the variant hinge region can be at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to SEQ ID NO: 3.
  • the variant hinge region can have a conservative modification as compared to SEQ ID NO: 3.
  • the variant hinge region can have a point mutation, an insertion, a deletion, a truncation, a fusion partner, or a combination thereof as compared to SEQ ID NO: 3.
  • the variant hinge region can have one, two, or three of SEQ ID NOs: 6-8, or has SEQ ID NO: 9 or 10.
  • the variant hinge region can have one copy of a 17-mer sequence (e.g., SEQ ID NO: 6) and three copies of a 15 mer sequence (e.g., SEQ ID NO: 7 or 8). In that case, at least one of the 17-mer or 15-mer sequence can be selected the group consisting of SEQ ID NOs: 6-8.
  • the hinge region is longer than that of IgGl hinge region (19 amino acid residues) and at least 20 (e.g., at least 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, 70, 80, 90, or 100) amino acid residues in length.
  • the variant hinge region can comprise the sequence of SEQ ID NO: 3, where 9 of the 11 cysteine are changed to serines (S).
  • the first antigen-binding site is derived from a first parent antibody, the CHI and CL domains of which are swapped in the first antigen-binding site. That is, each of the heavy chains is a fusion polypeptide having the VH-CL domains; and each of the light chain is fusion polypeptide having the VL-CH1 domains.
  • the second antigen-binding site can also be derived from a second parent antibody, the CHI and CL domains of which are swapped in the second antigen-binding site. As disclosed in FIG. 1 A here, such swapping of the CHI domain with the CL domain for the one but not for the other parental antibody allows one to pair correct light chains with the corresponding heavy chain.
  • the first antigen-binding site has substantially the same antigen binding activity as the first parent antibody, and similarly, the second antigen- binding site has substantially the same antigen binding activity as the second parent antibody
  • the first epitope and the second epitope mentioned above are non-overlapping epitopes on the envelope protein of HIV- 1.
  • the first epitope or the second epitope can be located in the CD4-binding site, the V3 loop, the V1/V2 loop, the gpl20/gp41 interface, or the MPER region of the envelope protein.
  • the first antigen-binding site or the second antigen-binding site can be derived from a parent bNAb selected from the group consisting of 3BNC117, 8ANC131, CH103, 10-1074, PGT121, PGT128, PGT135, PG16, PGT145, PGDM1400, PGT151, 8ANC195, and 10E8.
  • the first antigen-binding site and the second antigen-binding site can be derived from a pair of different parent bNAbs and show synergy as compared to the parent pair.
  • the first antigen-binding site and the second antigen-binding site can be derived from a pair of different parent bNAbs and show synergy as compared to the parent pair.
  • bispecific antibodies derived from the following pairs exhibit synergy on the neutralization activity against a small virus panel (wherein a positive number indicates synergy):
  • the second antigen-binding site is from the group consisting of PGT121, PGT135, PG16, 8ANC195, and 10E8;
  • the second antigen-binding site is from the group consisting of PGT128, PGT135, PGT145, PGDM1400 PGT151,
  • the second antigen-binding site is from the group consisting of PGT135, PGT145, PGT151, and 10E8;
  • the second antigen-binding site is from the group consisting of PG16, PGT151, and 8ANC195;
  • the second antigen-binding site is from the group consisting of 3BNC117, PG16, PGT151, and 10E8;
  • the first antigen-binding site is from PGT128, the second antigen-binding site is from the group consisting of 8ANC131, PG16, PGT151, and 8ANC195;
  • the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, CH103, PG16, PGT145, PGDM1400, PGT151, and 10E8;
  • the second antigen-binding site is from the group consisting of 3BNC117, 10-1074, PGT121, PGT128, PGT135, PGT151, 8ANC195, and 10E8;
  • the second antigen-binding site is from the group consisting of 8ANC131, CH103, PGT135, PGT151, 8ANC195, and
  • the second antigen-binding site is from the group consisting of 8ANC131, PGT135, PGT151, and 10E8;
  • the second antigen-binding site is from the group consisting of 8ANC131, CH103, 10-1074, PGT121, PGT128, PGT135, PG16, PGT145, PGDM1400, 8ANC195, and 10E8;
  • the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, 10-1074, PGT128, PG16, PGT145, PGT151, and 10E8; or
  • the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, CH103, PGT121, PGT135, PG16, PGT145, PGDM1400, PGT151, and 8ANC195.
  • Bispecific antibodies derived from the other pairs shown in FIG 3B and disclosed herein are also within the scope of this invention as they can show synergy for other virus strains.
  • the first antigen-binding site is derived from 3BNC117 and the second antigen-binding site is derived from 10-1074 or PGT 135, respectively.
  • the bispecific antibody described above can be used in a method of treating or preventing or controlling an HIV infection.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody.
  • the invention also provides a pharmaceutical composition comprising (i) the bispecific antibody described above, or a bispecific antigen-binding fragment thereof, and (ii) a pharmaceutically acceptable carrier.
  • the invention provides a kit comprising a pharmaceutically acceptable dose unit of a pharmaceutically effective amount of at least one bispecific antibody described above or a bispecific antigen-binding fragment thereof.
  • the invention provides a method of detecting an HIV infection in a subject.
  • the method includes contacting a biological sample from the subject with the antibody described above or bispecific antigen binding fragment thereof under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the sample from the subject.
  • the presence of the immune complex in the sample from the subject indicates that the subject has an HIV infection.
  • an isolated polypeptide comprising one or more of the following: a first sequence that is at least 75% identical to SEQ ID NO: 6 and has a non- cysteine residue (e.g., a serine) at position 13 or 16 or both of SEQ ID NO: 6; a second sequence that is at least 75% identical to SEQ ID NO: 7 and has a non-cysteine residue (e.g., a serine) at one or more of the positions 5, 1 1, and 14 of SEQ ID NO: 7, a third sequence that is at least 75% identical to SEQ ID NO: 8 and has a non-cysteine residue (e.g., a serine) at position 5 of SEQ ID NO: 8, a fourth sequence that is at least 75% identical to SEQ ID NO: 9 and has a non-cysteine residue (e.g., a serine) at one or more of positions 13, 16, 22, 28, 31, and 37 of SEQ ID NO: 9, and a fifth sequence that is at least 75%) identical to SEQ.
  • the isolated polypeptide can have different combination of the three sequences.
  • the polypeptide is at least 60%> (e.g., any number between 60%> and 100%>, inclusive, e.g., 60%, 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to SEQ ID NO: 3, 8 or 9.
  • the first sequence can be 17 or more mer in length.
  • the second and third sequence can be 15 mer or more in length.
  • This polypeptide can be used as a hinge region for the antibody described herein.
  • the polypeptide generally has the same length as or is longer than a reference sequence (e.g., one of SEQ ID Nos: 1-10).
  • nucleic acid comprising a sequence encoding a chain of the isolated antibody or antigen-binding fragment thereof or a polypeptide describe above.
  • the nucleic acid can be used to express a polypeptide, a chain of the antibody, or antigen-binding fragment of this invention, or the antibody or fragment.
  • suitable regulatory sequences for this purpose, one can operatively link the nucleic acid to suitable regulatory sequences to generate an expression vector.
  • a cultured host cell comprising the vector and a method for producing a polypeptide, the method comprising culturing the host cell under conditions in which the nucleic acid molecule is expressed.
  • bispecific anti-HIV antibodies In addition to bispecific anti-HIV antibodies, other bispecific antibodies such as anti-tumor bispecific antibodies and related methods are also provided.
  • FIGs. 1A, IB, 1C, ID, IE, IF and 1G are an overview of bispecific antibody generation and neutralization activity of IgGl bispecific neutralizing antibodies (biNAbs).
  • FIG. 1A Overview of the strategy for generating biNAbs to ensure proper heavy-light chain pairing and heterodimerization.
  • FIG. IB Comparison of the in vitro neutralization activity of wild-type and CrossMab variants of 10-1074 mAbs.
  • FIG. 1C - FIG. 1G In vitro neutralization breadth and potency plots of different combinations of biNAbs against an extended multiclade virus panel. Neutralization activity of their respective parental mAbs was included for comparison. All biNAb combinations exhibited marked reduction in neutralization potency compared to the activity of a mix of their parental mAbs.
  • FIGs. 2A, 2B and 2C show generation and characterization of biNAb hinge domain variants.
  • FIG.2A Schematic representation and primary amino acid sequence of the hinge domain of IgGl, IgG3 and the generated variant, IgG3C- (SEQ ID NOs: 1-3). Disulphide bonds and Cys residues are depicted in red.
  • IgGl hinge domain variants of 10- 1074 mAb were generated by switching the IgGl hinge region with the IgG3 or IgG3C- hinge.
  • FIG. 2A Schematic representation and primary amino acid sequence of the hinge domain of IgGl, IgG3 and the generated variant, IgG3C- (SEQ ID NOs: 1-3). Disulphide bonds and Cys residues are depicted in red.
  • FIGs. 3A, 3B and 3C show neutralization activity of hinge domain engineered (IgG3C-) biNAbs.
  • a panel of mAbs were selected targeting different epitopes on Env. (FIG. 3A) epitope mapping on the surface of the HIV-1 Env trimer and biNAb combinations (encompassing the IgG3C- hinge variant) with non-overlapping epitope specificities were generated.
  • FIG. 3B In vitro neutralization activity against a cross-clade virus panel was assessed and combinations with variable degree of synergy were identified (FIG. 3B) grid showing the fold change (log) in activity of biNAb over the respective parental mAbs; (FIG. 3C) IC50 titers of example biNAb combinations exhibiting synergistic, neutral or inhibitory activity.
  • FIGs. 4 A, 4B, 4C, 4D, 4E, 4F and 4G show in vitro neutralization potency and in vivo protective activity of IgG3C- hinge biNAbs.
  • IgG3C- hinge variants of PGT151/10-1074 FIG. 4A
  • 8ANC195/PGT128 FIG. 4B
  • 3BNC117/PGT135 FIG. 4C
  • FIGs. 5A, 5B, 5C and 5D show characterization of 3BNC117/10-1074 biNAbs.
  • FIG. 5 A Binding specificity for gpl40 of wild-type and CrossMab variant of 10-1074 was assessed by ELISA using recombinant gpl40.
  • FIG. 5B - FIG. 5D ELISA assays to determine dual specificity of the 3BNC117/10-1074 biAb.
  • FIG. 5B 3BNC117/10-1074 biNAb, 3BNC117 and 10-1074 mAbs were immobilized to gpl40-coated microtiter plates and detected using Fc domain- or light chain (kappa or lambda) subclass-specific secondary IgG.
  • BiAb was detected with both the anti-kappa and anti-lambda secondary antibodies, whereas 3BNC117 and 10-1074 only with anti -kappa or anti -lambda, respectively.
  • FIG. 5C Competition ELISA with increasing concentrations of each of the parental mAbs or a mixture of the two mAbs to determine dual specificity of the bispecific mAb.
  • FIG. 5D Epitope-specific ELISA using a CD4bs antigen (2-CC Core) for capture and an anti-lambda detection antibody to confirm bispecific activity.
  • FIGs. 6A and 6B show size exclusion chromatography (SEC) and thermostability of hinge domain variants of 10-1074. Hinge domain variants of 10-1074 were analyzed (FIG. 6A) by SEC to assess for protein aggregation and (FIG. 6B) by thermal shift protein assay to determine protein Tm.
  • SEC size exclusion chromatography
  • FIGs. 7A, 7B, 7C, 7D, 7E and 7F show comparison of the neutralization activity of IgGl and IgG3C- hinge variants of anti-HIV-1 Env mAbs.
  • FIG. 7A 3BNC117; FIG. 7B: 10-1074;
  • FIG. 7C PGT128;
  • FIG. 7D PGT135;
  • FIG. 7E PGT151;
  • FIG. 7F 8ANC195
  • FIGs. 8 A and 8B show crystal structure of the PGT135 and 3BNC117 Fabs bound to the Env trimer.
  • FIG. 8 A Side and
  • FIG. 8B top view of 3BNC117 (cyan; 4JPV) and PGT135 (red; 4JM2) Fabs bound to the Env trimer (4NCO).
  • the distance between the ends of the two Fabs was calculated to be 67A, suggesting that the IgG3C- hinge variant might facilitate bivalent, intra-trimeric interactions of the 3BNC117/PGT136 biNAb.
  • FIGs. 9A, 9B, and 9C show in vivo IgG half-life and mutation analysis of biNAb- treated HIV-1 infected humanized mice.
  • HIV-1- infected humanized mice were treated for 4 weeks with either a mix of 3BNC117 + PGT135 IgG3C- mAbs or 3BNC117/PGT135 IgG3C- biNAb.
  • HIV-1 gpl20 from mice exhibiting viremia of >10 4 copies/ml were cloned and their sequences were analyzed.
  • FIGs. 10A and 10B show in vitro neutralization activity (IC50 titers ⁇ g/ml)) of 3BNC117 + PGT135 bNAb mix or 3BNC117/PGT135 biNAb expressed with the following hinge domain structures: IgGl, IgG3 and IgG3C-.
  • 3BNC117/PGT135 IgG3C- biNAb exhibited significantly improved neutralization activity (**p ⁇ 0.001) compared to IgGl or IgG3 biNAb hinge variants.
  • IC50 titer ⁇ g/ml) results are presented as geometric mean ⁇ 95%CI.
  • FIGs. 11A and 11B show IC50 and IC80 titers (ng/ml) of 3BNC117/PGT135
  • IgG3C- biNAbs with variable hinge domain length Shorter variants of IgG3C- ("Full length") were generated by deleting either one ("-15mer”) or two ("-2xl5mer”) of the three 15-mer repeats.
  • FIG 11B also shows comparison of the neutralization activity between 3BNC117/PGT135 IgG3C- biNAb (full length) and shortened hinge domain variants revealed that enhanced neutralization activity is correlated with hinge domain length.
  • IC50 titer ⁇ g/ml) results are presented as geometric mean ⁇ 95%CI. *p ⁇ 0.05; **p ⁇ 0.001.
  • FIG 12 shows determination of half-life of 3BNC117/PGT135 IgG3C- biNAb in Rhesus macaques.
  • FIG. 13 A and 13B show comparison of the neutralization activity of the
  • FIG. 13 A Neutralization breadth and potency plot comparing the activity of the 3BNC117/PGT135 IgG3C- biNAb to various bNAbs targeting distinct epitopes on HIV-1 Env.
  • FIG. 13B Comparison of the neutralization potency of the 3BNC117/PGT135 IgG3C- biNAb with single bNAbs and bNAb combinations (2-4 bNAbs). Neutralization data for these bNAbs and biNAb combinations have been previously reported in Kong et al., (2015). Comparative analysis was performed using a panel of 115 viruses, for which neutralization data were available both for the biNAb and the bNAb combinations. DETAILED DESCRIPTION OF THE INVENTION
  • This invention provides novel bispecific anti-HIV bNAbs. As disclosed herein, to overcome the above-discussed limitations associated with virus escape mechanisms following antibody treatment, a panel of bispecific anti-HIV bNAbs were generated and characterized.
  • bNAbs Broadly neutralizing monoclonal antibodies (bNAbs) against the envelope glycoprotein of HIV-1 (Env)
  • Env envelope glycoprotein of HIV-1
  • Env can suppress viremia in animal models of HIV-1 and humans (Barouch, D. H. et al., Nature 503, 224-228, doi: 10.1038/nature 12744 (2013); Bournazos, S.
  • biNAbs bispecific anti-Env neutralizing antibodies
  • Synergistic activity of biNAbs was achieved by engineering the hinge domain of IgGl through the use of variants based on the IgG3 hinge (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997)) to increase Fab domain flexibility for hetero-bivalent binding to the Env trimer.
  • hinge domain variants Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1 -infected humanized mice.
  • This invention provides novel strategies for generating biNAbs with improved in vitro and in vivo activity through hinge domain engineering. Such biNAbs exhibit remarkable neutralization breadth and potency and appear to be ideal candidate molecules for the prevention and treatment of HIV-1 infection.
  • Bispecific neutralizing antibodies represent an attractive therapeutic strategy for the prevention and treatment of HIV-1 infection, given their unique advantages over conventional, monospecific antibodies. Indeed, biNAbs can overcome virus escape issues associated with the use of conventional bNAbs (Halper-Stromberg, A. et al, Cell 158, 989-999, doi: 10.1016/j .cell.2014.07.043 (2014); Horwitz, J. A. et al, Proc NatlAcadSci USA 110, 16538-16543, doi: 10.1073/pnas. l315295110 (2013); Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)), providing a compelling platform for the development of single therapeutic molecules that would combine the breadth and potency of two bNAbs for effective control of HIV-1 infection.
  • a key immune evasion mechanism of HIV-1 against host antibody responses is the remarkably low density of Env molecules on the viral surface, as well as the unique Env trimeric architecture, which preclude high-avidity bivalent interactions of IgG (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010).). It is therefore likely that conventional biNAbs would exhibit predominantly monovalent binding to their respective epitopes, possibly accounting for the lack of additive, synergistic activity. Indeed, given the relative rigidity and the short length of the hinge domain of IgGl, concurrent binding of the two Fab arms within the same Env trimer is largely restricted.
  • IgG3 encompasses an exceptionally long and flexible hinge domain, with distinct structural and functional characteristics (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997) and Roux, K. H., et al, J Immunol 761, 4083-4090 (1998)) (FIG. 2A). It is comprised of a 17-mer amino acid sequence followed by three 15 mer repeats that are highly homologous to the IgGl hinge structure and represent genomic duplication events of the ancestral hinge-encoding exon, conserved among all other IgG subclasses (Roux, K.
  • IgG3C- IgG3-based hinge variant
  • C cysteine residues
  • S serines
  • hinge domain variants of 10-1074 are shown in the examples below.
  • IgGl were expressed as chimeric molecules, in which the wild-type hinge domain (IgGl) was replaced with that of either IgG3 or the IgG3C- variant.
  • the hinge domain all other domains of the constant region (CHI, CH2, CH3) were of the IgGl subclass to preserve the effector function and half-life of wild-type IgGl .
  • hinge domain variants of 10-1074 demonstrated comparable binding affinity to the different classes of human and mouse FcyRs, suggesting a minimal role for the hinge region in Fc-FcyR interactions (Extended Data Table 1).
  • no differences among the hinge domain variants were noted in terms of protein stability and in vivo pharmacokinetics (FIG. 2B, and FIG. 6).
  • This invention accordingly encompasses a bispecific antibody comprising (i) a first antigen-binding site that binds to a first epitope and (ii) a second antigen-binding site that binds to a second epitope.
  • the first antigen-binding site comprises a first heavy chain and a first light chain while the second antigen-binding site comprises a second heavy chain and a second light chain.
  • the first heavy chain and the second heavy chain comprises a variant hinge region that (a) is derived from a wild type IgG3 hinge region and (b) has fewer CH-1 proximal cysteine residues than the wild type IgG3 hinge region as compared to a reference antibody containing the wild type IgG3 hinge region. Shown below are exemplary hinge sequences and variants thereof. Variants of these hinge sequences with conservative modifications can also be used. SEQ Name Sequences Note
  • EPKSCDTPPPCPRCP EPKSCDTPPPCPRCP EPKSCDTPPPCPRCP APEL
  • IgG3 hinge region ELKTPLGDTTHTCPRCP This is the same as aa 1-17 of 17-mer wild type SEQ ID NO: 2. It is a part of
  • EPKSCDTPPPCPRCP This correspond to aa 18-32 of 15-mer wild type SEQ ID NO: 2. It is a part of
  • IgG3 C-hinge ELKTPLGDTTHTSPRSP This is a mutant version of SEQ region 17-mer ID NO: 4. It is a part of SEQ ID mutant NO: 3.
  • mutant B It is a part of SEQ ID NO: 3.
  • EPKSSDTPPPCPRCP APEL 11B EPKSSDTPPPCPRCP APEL 11B.
  • heterodimerization of the heavy chains of the two mAbs was achieved by introducing mutations in the CH3 domain that alter the physical and chemical properties of the two heavy chains favoring heterodimerization. These mutations have no impact on the capacity of the Fc domain to interact with FcyRs and FcRn, having thereby no effect on Fc effector function and IgG half-life.
  • Pairing of the correct heavy chain with the respective light chain was achieved by swapping the CHI domain with CL for one of the parental bNAbs, while maintaining the wild-type architecture for the other bA b. These approaches typically yielded >90% heterodimers, without evidence for protein aggregation ( ⁇ 10% IgG multimers).
  • hinge domain-engineered bNAbs are based on the naturally occurring hinge domain of human IgG3.
  • Human IgG3 encompasses an exceptionally long and flexible hinge domain compared to other IgG subclasses, which has been previously suggested to contribute to the improved effector activity of this IgG subclass.
  • IgG3 C- an "open" IgG3 hinge variant
  • biNAbs with non-overlapping epitope specificities were developed and characterized to identify particular biNAb combinations that would demonstrate potent and synergistic neutralization activity.
  • the inventors Given the significance of the FcyR-mediated pathways in the in vivo protective activity of anti-HIV-1 bNAbs (Bournazos et al, 2014, Cell 158, 1243-1253; Halper-Stromberg et al, 2014 Cell 158, 989-999; Hessell et al, 2007, Nature 449, 101-104), the inventors generated biNAbs with the wild-type IgG structure, while avoiding irregular, non-physiological architectures used in the past (Spiess et al, 2015, Mol Immunol 67, 95-106).
  • biNAb would have the capacity to interact with FcyRs and exhibit long and stable in vivo pharmacokinetics, with minimal immunogenicity potential. Indeed, all the generated biNAb variants exhibited identical in vivo half-life and affinity for the different classes of FcyRs. Attempts to generate anti-HIV-1 Env biNAbs based on the IgGl structure were generally characterized by lower neutralization potency compared to their corresponding parental bNAbs, irrespective of their epitope specificity.
  • hinge domain engineered biNAbs were generated with improved Fab domain flexibility, based on the naturally-occurring hinge domain of IgG3, which is characterized by a uniquely long and flexible structure (Roux et al, 1998, J Immunol 161, 4083-4090.; Roux et al., 1997, J Immunol 159, 3372-3382). This approach incorporates minimal changes to the overall IgG structure, while it achieves significant enhancement in the neutralization breadth and potency.
  • 3BNC117/PGT135 showed evidence for synergistic activity, surpassing the potency of both parental bNAbs (3BNC117 and PGT135). Indeed, assessment of its neutralization potency indicated that for the vast majority of the tested viruses, the 3BNC117/PGT135 biNAb exhibits lower IC50 and IC80 titers, and for over a third of the tested viruses, the improvement in the neutralization potency exceed 10-fold compared to 3BNC117 and PGT135 bNAbs. Its enhanced activity was largely attributed to the hinge length and flexibility, as hinge domain variants with shorter length or decreased flexibility also exhibited reduced neutralization breadth and potency.
  • the hinge-engineered 3BNC117/PGT135 biNAb represents one of the most potent anti-HIV-1 bNAbs developed to date, exhibiting high neutralization breadth and potency, as well as improved in vivo activity.
  • its neutralization breadth and potency was compared to several bNAbs targeting distinct epitopes on gpl40.
  • the 3BNC117/PGT135 biNAb displays increased neutralization potency, compared to the most potent bNAbs so far characterized, including VRC07, PGT121, and PG9.
  • bispecific anti-HIV-1 antibodies offers significant advantages over conventional monoclonal antibodies, as they combine the breadth and potency of two broadly neutralizing antibodies and overcome viral escape mechanisms associated with the use of single antibody preparations.
  • hinge domain variants were generated based on the IgG3 scaffold to optimize Fab domain flexibility, favoring intra-molecular Env interactions. Hinge-modified bispecific antibodies exhibited improved potency, with evidence for enhanced in vitro and in vivo activity.
  • a panel of IgG3 C- variants of bispecific bNAbs was generated and their in vitro neutralization activity was assessed against a small panel of HIV isolates, using a standardized TZM-bl assay.
  • As control the activity of IgG3 C- monospecific bNAbs were assessed and no difference in their activity was evident when compared to their IgGl counterparts.
  • comparison of the activity of IgG3 C- bispecific bNAbs revealed that particular bispecific combinations exhibit significant synergistic activity compared to the corresponding IgG3 C- parental bNAbs.
  • the present invention describes an approach for the generation of anti- HIV-1 Env biNAbs with physiological IgG architecture, FcyR binding profile and pharmacokinetic properties.
  • biNAbs with hinge domain engineered structures exhibit potent neutralization activity with improved breadth and potency and enhanced in vivo activity.
  • the bispecific antibodies of the present invention can be made using or derived from the chains of any isolated antibodies ⁇ i.e., parental antibodies), in particular monoclonal antibodies such as human monoclonal antibodies that bind to different antigens or epitopes.
  • monoclonal antibodies such as human monoclonal antibodies that bind to different antigens or epitopes.
  • the antibodies are human antibodies, although the antibodies can also be, for example, murine antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • Monoclonal antibody techniques allow for the production of specifically binding agents in the form of specifically binding monoclonal antibodies or fragments thereof.
  • monoclonal antibodies, or fragments thereof For creating monoclonal antibodies, or fragments thereof, one can use conventional hybridoma techniques.
  • monoclonal antibodies, or fragments thereof can be obtained by the use of phage libraries of scFv (single chain variable region), specifically human scFv (see e.g. U.S. Pat. No. 5,885,793, WO 92/01047, and WO 99/06587).
  • anti-HIV bNAbs antibodies refer to a class of neutralizing antibodies that neutralize multiple HIV-1 viral strains.
  • Various bNAbs are known in the art and can be used in this invention. Examples include but are not limited to those described in U.S. Patent NO. 8673307, WO2014063059, WO2012158948, WO2015/117008, and PCT/US2015/41272, including antibodies 3BNC117, 3BNC60, 12A12, 12A21, NIH45- 46, bANC131, 8ANC134, IB2530, INC9, 8ANC195.
  • the anti-HIV bNAbs of this invention encompass the antigenic specificity of two potent bNAbs targeting distinct, non-overlapping epitopes on the HIV-1 Env necessary for viral fitness.
  • epitopes include: (i) the CD4 binding site, which is targeted by the 3BNC117, 8ANC131 and CHI 03 mAbs; (ii) the V3 region, which is recognized by the 10-1074, PGT121, PGT128, and PGT135 mAbs in a glycan dependent or independent fashion; (iii) the Vl/2 region, recognized by the PG16, PGT145 and PGDM1400 mAbs, (iv) the membrane proximal region (MPER), targeted by the 10E8 mAb, and (v) the gpl20/gp41 interface, which is recognized non-competitively by the PGT151 and 8ANC195 mAbs.
  • VH heavy chain variable regions
  • VH heavy chain variable regions
  • GQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTIS GVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLGQPKA AP
  • YQYKPGQSPRLVI FETYSKIAAFPARFVASGSGTEFTLT INNMQSEDVAVYYCQQYEEWPRTFGQGTKVDIKRTVAAP PG16 VH QEQLVESGGGWQPGGSLRLSCLASGFTFHKYGMHWVRQ 25 APGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNT LYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDG YYNYHYMDVWGKGTTVTVSSAS
  • VPGQGLQWMGWISHEGDKKVIVERFKAKVTIDWDRSTNT AYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGALN WAVDVDYLSNLEFWGQGTAVTVSSAS
  • HIV bNAbs but also other bispecific therapeutic antibodies.
  • a number of therapeutic antibodies directed against cell surface molecules and/or their ligands are known which can be used for the selection and construction of tailor-made specific recognition binding moiety in the bispecific antibodies of this invention. Examples include
  • bispecific binding molecules/bispecific antibodies disclosed herein can be used in the preparation of medicaments for the treatment of e.g. an oncologic disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune disease, a metabolic (e.g., endocrine) disease, or a neurological (e.g. neurodegenerative) disease.
  • an oncologic disease e.g. an oncologic disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune disease, a metabolic (e.g., endocrine) disease, or a neurological (e.g. neurodegenerative) disease.
  • Non-limiting examples of these diseases are Alzheimer's disease, non- Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T-cell lymphomas and leukemias, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas (such as carcinomas of the oral cavity, gastrointestinal tract, colon, stomach, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes), melanomas, sarcomas, gliomas, and skin cancers, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham
  • cell surface markers and their ligands are known.
  • cancer cells have been reported to express at least one of the following cell surface markers and or ligands, including but not limited to, carbonic anhydrase IX, alpha-fetoprotein, alpha- ctinin-4, A3 (antigen specific for A33 antibody), ART -4, B7, Ba-733, BAGE, BrE3- antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD 1, CDla, CD2, CD3, CD4, CDS, CD8, CD1-1A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD
  • antibodies recognizing such specific cell surface receptors or their ligands can be used for specific and selective recognition binding moieties in the bispecific antibodies of this invention, targeting and binding to a number/multitude of cell surface markers or ligands that are associated with a disease.
  • bispecific antibodies are engineered that simultaneously bind to a cytotoxic cell ⁇ e.g., using a receptor like CD3) and a target like a tumor cell to be destroyed. See, e.g., Mueller et al, 2010). Biodrugs 24 (2): 89-98 and Chames et al, (2009). MAbs 1 (6): 539-547. Accordingly, the invention disclosed herein can also be used in cancer immunotherapy.
  • bispecific antibodies are used to target tumor-associated antigens (TAAs), such as those reported in Herberman, "Immunodiagnosis of Cancer", in Fleisher ed., "The Clinical Biochemistry of Cancer", page 347 (American Association of Clinical Chemists, 1979) and in U.S. Pat. No. 4,150, 149; U.S. Pat. No. 4,361,544; and U.S. Pat. No. 4,444,744.
  • TAAs tumor-associated antigens
  • targeted antigens may be selected from the group consisting of CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CXCR4, B7, MUCl or la, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product (e.g., c-met or PLAGL2), CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
  • Antibodies against the above-mentioned antigens can be used as the binding sites or moieties to make bispecific antibodies of this invention.
  • a number of bispecific antibodies can be made against two different targets.
  • antigen pairs examples include CD19/CD3, BCMA/CD3, different antigens of the HER family in combination (EGFR, HER2, HER3), IL17RA/IL7R, IL-6/IL-23, IL- l-p/IL-8, IL-6 or IL-6R/IL-21 or IL-21R, ANG2/VEGF, VEGF/PDGFR-beta, Vascular Endothelial Growth Factor (VEGF) acceptor 2/CD3, PSMA/CD3, EPCAM/CD3, combinations of antigens selected from a group consisting of VEGFR-l, VEGFR-2, VEGFR-3, FLT3, c-FMS/CSFIR, RET, c-Met, EGFR, Her2/neu, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrin and MMPs with a water-soluble ligand is selected from the group consisting of VEGF, EGF, PIGF, PDGF, HGF,
  • bispecific antibodies can have (i) a first specificity directed to a glycoepitope of an antigen selected from the group consisting of Lewis x-, Lewis b- and Lewis y-structures, Globo H-structures, KH1, Tn-antigen, TF-antigen and carbohydrate structures of Mucins, CD44, glycolipids and glycosphingolipids, such as Gg3, Gb3, GD3, GD2, Gb5, Gml, Gm2, and sialyltetraosylceramide and (ii) a second specificity directed to an ErbB receptor tyrosine kinase selected from the group consisting of EGFR, HER2, HER3 and HER4, GD2 in combination with a second antigen binding site is associated with an immunological cell chosen from the group consisting of T- lymphocytes K cell, B-lymphocytes, dendritic cells, monocytes, macrophages, neutrophils, mesenchymal stem cells,
  • a monospecific antibody can be joined together with another using the method disclosed herein to make bispecific antibodies.
  • Another using the method disclosed herein to make bispecific antibodies By using already available specific therapeutic binding entities, such as those therapeutic antibodies described above, one can generate a desired combination.
  • this tailor-made generation of bispecific therapeutics by combining two single therapeutic molecules for simultaneous targeting and binding to two different epitopes, an additive/synergistic effect can be expected in comparison to the single therapeutic molecules.
  • an “antigen” refers to a substance that elicits an immunological reaction or binds to the products of that reaction.
  • epitopope refers to the region of an antigen to which an antibody or T cell binds.
  • antibody as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), and antibody fragments.
  • antibody as used in any context within this specification is meant to include, but not be limited to, any specific binding member, immunoglobulin class and/or isotype (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant fragment or specific binding member thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity).
  • immunoglobulin class and/or isotype e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM
  • biologically relevant fragment or specific binding member thereof including but not limited to Fab, F(ab')2, Fv, and scFv
  • an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • a heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CHI, CH2 and CH3).
  • a light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the variable regions of both the heavy and light chains comprise framework regions (FWR) and complementarity determining regions (CDR).
  • CDR1, CDR2 and CDR3 represent hypervariable regions and are arranged from NH2 terminus to the COOH terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen while, depending of the isotype, the constant region(s) may mediate the binding of the immunoglobulin to host tissues or factors.
  • antibody as used herein are chimeric antibodies, humanized antibodies, and recombinant antibodies, human antibodies generated from a transgenic non-human animal, as well as antibodies selected from libraries using enrichment technologies available to the artisan.
  • variable refers to the fact that certain segments of the variable (V) domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 110-amino acid span of the variable regions.
  • the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each 9-12 amino acids long.
  • the variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a “complementarity determining region” (“CDR").
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • polyclonal antibody refers to preparations that include different antibodies directed against different determinants ("epitopes").
  • the monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with, or homologous to, corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, for example, U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • Chimeric antibodies include antibodies having one or more human antigen binding sequences (for example, CDRs) and containing one or more sequences derived from a non-human antibody, for example, an FR or C region sequence.
  • chimeric antibodies included herein are those comprising a human variable region antigen binding sequence of one antibody class or subclass and another sequence, for example, FR or C region sequence, derived from another antibody class or subclass.
  • a “humanized antibody” generally is considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues often are referred to as "import” residues, which typically are taken from an "import” variable region. Humanization may be performed following the method of Winter and co-workers (see, for example, Jones et al, Nature 321 :522-525 (1986); Reichmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567), where substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species.
  • antibody fragment comprises a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see, for example, U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Fv is the minimum antibody fragment that contains a complete antigen- recognition and antigen-binding site. This fragment contains a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv (“sFv” or “scFv”) are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • sFv Single-chain Fv
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • DAbs Domain antibodies
  • VH and VL immunoglobulins
  • DAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL, respectively). They are highly expressed in microbial cell culture, show favorable biophysical properties including, for example, but not limited to, solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as, for example, phage display.
  • DAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities. Examples of this technology have been described in, for example, W09425591 for antibodies derived from Camelidae heavy chain Ig, as well in US20030130496 describing the isolation of single domain fully human antibodies from phage libraries.
  • Fv and sFv are the only species with intact combining sites that are devoid of constant regions. Thus, they are suitable for reduced nonspecific binding during in vivo use.
  • sFv fusion proteins can be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of a sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra.
  • the antibody fragment also can be a "linear antibody", for example, as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.
  • antibodies of the described invention are bispecific and can bind to two different epitopes of a single antigen.
  • Other such antibodies can combine a first antigen binding site with a binding site for a second antigen.
  • an anti- HIV arm can be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (for example, CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD 16), so as to focus and localize cellular defense mechanisms to the infected cell.
  • a triggering molecule on a leukocyte such as a T-cell receptor molecule (for example, CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32) and F
  • Bispecific antibodies also can be used to localize cytotoxic agents to infected cells.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (for example, F(ab')2 bispecific antibodies). See for example, WO 96/16673, U.S. Pat. No. 5,837,234, WO98/02463, U.S. Pat. No. 5,821,337, and Mouquet et al, Nature. 467, 591-5 (2010).
  • bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar procedures are disclosed in, for example, WO 93/08829, Traunecker et al., EMBO J., 10:3655-3659 (1991) and see also; Mouquet et al, Nature. 467, 591-5 (2010). Techniques for generating bispecific antibodies from antibody fragments also have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. See Brennan et al, Science, 229: 81 (1985).
  • the parent antibodies described in the invention can be produced using conventional hybridoma technology or made recombinantly using vectors and methods available in the art.
  • Human antibodies also can be generated by in vitro activated B cells (see, for example, U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • General methods in molecular genetics and genetic engineering useful in the present invention are described in the current editions of Molecular Cloning: A Laboratory Manual (Sambrook, et al., Molecular Cloning: A Laboratory Manual (Fourth Edition) Cold Spring Harbor Lab. press, 2012), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991.
  • Human antibodies also can be produced in transgenic animals (for example, mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • transgenic animals for example, mice
  • JH antibody heavy-chain joining region
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • Phage display technology is known in the art (e.g., see technology from Cambridge Antibody Technology (CAT)) as disclosed in U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6, 140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593, 081, as well as other U.S. family members, or applications which rely on priority filing GB 9206318, filed 24 May 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14: 309- 314).
  • Single chain antibodies may also be designed and constructed using available recombinant DNA technology, such as a DNA amplification method (e.g., PCR), or possibly by using a respective hybridoma cDNA as a template.
  • variants of the sequences recited in the application also are included within the scope of the invention.
  • Further variants of the antibody sequences having improved affinity can be obtained using methods known in the art and are included within the scope of the invention.
  • amino acid substitutions can be used to obtain antibodies with further improved affinity.
  • codon optimization of the nucleotide sequence can be used to improve the efficiency of translation in expression systems for the production of the antibody.
  • an antibody of the invention comprises a heavy chain variable region comprising CDRl, CDR2 and CDR3 sequences, and a light chain variable region comprising CDRl, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the preferred antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of neutralizing multiple HIV-1 viral strains.
  • an antibody of the invention can comprise a hinge region of the preferred antibodies described herein, e.g., SEQ ID NO: 3, a section thereof, or conservative modifications thereof.
  • a conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed in this invention refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity to of the parent peptide, polypeptide, or protein (such as those disclosed in this invention).
  • a conservative modification or functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to a parent (e.g., one of SEQ ID NOs: 1-36).
  • a parent e.g., one of SEQ ID NOs: 1-36.
  • hinge regions having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof, as well as heavy chains or antibodies having the variant hinge regions.
  • a variant hinge region can be a truncated form of IgG3 hinge region or IgG3 C-hinge region, such as one with fewer than three repeats (e.g., two or one copy) of the 15 mer or with repeats of a sequence shorter than the 15 mer or 17 mer mentioned above.
  • the hinge region is longer than that of IgGl hinge region (19 amino acid residues) and at least 20 (e.g., at least 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 70, 80, 90, or 100) amino acid residues in length.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs ⁇ e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).
  • conservative modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include
  • amino acids with basic side chains ⁇ e.g., lysine, arginine, histidine
  • acidic side chains ⁇ e.g., aspartic acid, glutamic acid
  • uncharged polar side chains ⁇ e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains ⁇ e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains ⁇ e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the variant hinge region described herein can have one or more conservative amino acid substitutions.
  • the hinge region of antibody molecules is a flexible domain that joins the Fab arms to the Fc piece.
  • the flexibility of the hinge region in IgG and IgA molecules allows the Fab arms to adopt a wide range of angles, permitting binding to epitopes spaced variable distances apart. Since the variant hinge region is to provide flexibility, but not antigen-binding specificity or other function such as binding to a receptor, one skilled in the art would appreciate that various positions of the hinge regions disclosed herein can have conservative amino acid substitutions without comprising the flexibility.
  • the antibody can be linked to one of a variety of nonproteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody also can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
  • bNAbs are significantly more effective than ART in blocking the establishment of the reservoir when given early in the infection.
  • One of the key differences between antibodies and ART is that antibodies can engage a variety of host immune effector pathways by way of their Fc receptors (Nimmerjahn et al., Nat Rev Immunol, 2008. 8(1): p. 34-47). Consistent with this important difference, the mechanism by which antibodies interfere with the establishment of the reservoir is dependent on their ability to bind to Fc receptors.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • An Fc receptor is a protein found on the surface of certain cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for the Fc region (fragment crystallizable region) of an antibody.
  • Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. It was also known in the art that the Fc region of an antibody ensures that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins.
  • FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsFR, for IgA as FcaR and so on.
  • FcyR Fc receptors for IgG antibodies
  • FcsFR FcsFR
  • IgA FcaR
  • Surface receptors for immunoglobulin G are present in two distinct classes-those that activate cells upon their crosslinking (“activation FcRs") and those that inhibit activation upon co-engagement (“inhibitory FcRs").
  • FcyRI CD64
  • FcyRII CD32
  • FcyRIII CDI6
  • FcylV FcylV
  • FcyRII displays high affinity for the antibody constant region and restricted isotype specificity
  • FcyRII and FcyRIII have low affinity for the Fc region of IgG but a broader isotype binding pattern (Ravetch and Kinet, 1991; Hulett and Hogarth, Adv Immunol 57, 1-127 (1994)).
  • FcyRrV is a recently identified receptor, conserved in all mammalian species with intermediate affinity and restricted subclass specificity (Mechetina et al., Immunogenetics 54, 463-468 (2002); Davis et al, Immunol Rev 190, 123-136 (2002); Nimmerjahn et al, Immunity 23, 41-51 (2005)).
  • Fc-receptors Functionally there are two different classes of Fc-receptors: the activation and the inhibitory receptors, which transmit their signals via immunoreceptor tyrosine based activation (ITAM) or inhibitory motifs (ITEVI), respectively (Ravetch, in Fundamental Immunology W. E. Paul, Ed. (Lippincott-Raven, Philadelphia, (2003); Ravetch and Lanier, Science 290, 84-89 (2000).
  • ITAM immunoreceptor tyrosine based activation
  • ITEVI inhibitory motifs
  • FcR is a native sequence human FcR.
  • FcR including human FcR, binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (an “activating receptor") and FcyRIIB (an "inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITFM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al, Immunomethods, 4, 25-34 (1994); and de Haas et al, J Lab Clin Med, 126, 330-41 (1995), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundamental Immunology, ed William Paul 5th Ed. each of which is incorporated herein by reference).
  • Fc fragment or "Fc region” is used to define a C- terminal region of an immunoglobulin heavy chain.
  • Fc region is the tail region of an antibody that interacts with Fc receptors and some proteins of the complement system.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • a native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • a variant Fc region as appreciated by one of ordinary skill in the art comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one "amino acid modification.”
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is important for Fc receptor-mediated activity.
  • the N-glycans attached to this site are predominantly core- fucosylated diantennary structures of the complex type.
  • small amounts of these N-glycans also bear bisecting GlcNAc and a-2,6 linked sialic acid residues.
  • the bNAb antibody of this invention can include antibody variable regions with the desired binding specificities (antibody-antigen combining sites) fused to immunoglobulin constant domain sequences.
  • the fusion can be with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy-chain constant region (CHI) containing the site for light chain bonding is present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell.
  • the above-described antibodies can be used in combination with one or more anti- retroviral agents for the treatment of HIV latency and/or infection. See, e.g., US 2010/0166806, US 2010/0324034, and US 2012/0203014, which are hereby incorporated in their entirety.
  • compositions according to the present invention may also be administered in combination with other agents to enhance the biological activity of such agents.
  • agents may include any one or more of the standard anti-HIV agents which are known in the art, including, but not limited to, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddl).
  • AZT azidothymidine
  • ddC dideoxycytidine
  • ddl dideoxyinosine
  • compositions in accordance to the invention include, for example, raltegravir, maraviroc, bestatin, human chorionic gonadotropin (hCG), levamisole, estrogen, efavirenz, etravirine, indomethacin, emtricitabine, tenofovir disoproxil fumarate, amprenavir, tipranavir, indinavir, ritonavir, darunavir, enfuvirtide, and gramicidin.
  • raltegravir maraviroc
  • bestatin human chorionic gonadotropin (hCG)
  • levamisole estrogen
  • efavirenz etravirine
  • indomethacin emtricitabine
  • tenofovir disoproxil fumarate amprenavir, tipranavir, indinavir, ritonavir, darunavir, enfuvirtide, and gramicidin.
  • a bispecific antibody of this invention can be further conjugated to one or more effector moieties, e.g. cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • effector moieties e.g. cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • the effector moiety can be a drug, including but not limited to a maytansinoid (see U.S. Pat. No. 5,208,020, U.S. Pat. No. 5,416,064, EP 0 425 235), an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF, see U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780,588, U.S. Pat. No. 7,498,298), a dolastatin, a calicheamicin or derivative thereof (see U.S. Pat. No. 5,712,374, U.S. Pat. No. 5,714,586, U.S. Pat. No.
  • a maytansinoid see U.S. Pat. No. 5,208,020, U.S. Pat. No. 5,416,064, EP 0 425 235
  • an auristatin such as monomethylauristatin drug moieties DE and
  • the effector moiety can be an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain,
  • the effector moiety can be a radioactive atom.
  • radioactive isotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Rel 88 , Sml 53 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc99 or 1123, or a spin label for nuclear magnetic resonance (MR) imaging (also known as magnetic resonance imaging, MRI), such as I 123 again, I 131 , In 111 , F 19 , C 13 , N 15 , O 17 , gadolinium, manganese or iron.
  • MR nuclear magnetic resonance
  • the present invention provides a composition comprising at least one bNAb mentioned above alone or in combination with one of the other active agent mentioned above and a pharmaceutically acceptable carrier.
  • the composition may include a plurality of the antibodies having the characteristics described herein in any combination and can further include antibodies neutralizing to HIV as are known in the art.
  • compositions can be a single or a combination of antibodies disclosed herein, which can be the same or different, in order to prophylactically or therapeutically treat the progression of various subtypes of HIV infection.
  • an antibody or active agent When an antibody or active agent is administered to an animal or a human, it can be combined with one or more pharmaceutically acceptable carriers, excipients or adjuvants as are known to one of ordinary skilled in the art.
  • mice for example, SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bone marrow-ablated BALB/c
  • PBMCs peripheral blood mononuclear cells
  • the simian immune deficiency virus (SrV)/monkey model can be employed, as can the feline immune deficiency virus (FIV)/cat model.
  • the pharmaceutical composition can contain other pharmaceuticals, in conjunction with a vector according to the invention, when used to therapeutically treat AIDS. These other pharmaceuticals can be used in their traditional fashion (i.e., as agents to treat HIV infection).
  • the present invention provides an antibody- based pharmaceutical composition comprising an effective amount of an isolated bNAb of the invention, or an affinity matured version, which provides a prophylactic or therapeutic treatment choice to reduce the latent reservoir and infection of the HIV virus.
  • the pharmaceutical compositions of the present invention may be formulated by any number of strategies known in the art (e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: In McNally, E.J., ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins.
  • a pharmaceutically acceptable composition suitable for patient administration will contain an effective amount of the bNAb antibody in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable temperature range.
  • the pharmaceutical compositions can also include, depending on the formulation desired, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination.
  • an excipient that is useful in the pharmaceutical composition or formulation of this invention is an amount that serves to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to a subject in need thereof. It may serve to dilute the antibody or other active agent to a concentration which provides the desired beneficial palliative or curative results while at the same time minimizing any adverse side effects that might occur from too high a concentration. It may also have a preservative effect. Thus, for an active ingredient having a high physiological activity, more of the excipient will be employed. On the other hand, for any active ingredient(s) that exhibit a lower physiological activity, a lesser quantity of the excipient will be employed.
  • bNAb antibodies and antibody compositions comprising at least one or a combination of the antibodies described herein, can be administered for the prophylactic and therapeutic treatment of HIV viral infection.
  • composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier.
  • pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • carrier as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include, but not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including, but not limited to, ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as, but not limited to, polyvinylpyrrolidone; amino acids such as, but not limited to, glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, glucose, mannose, or dextrins; chelating agents such as, but not limited to, EDTA; sugar alcohols such as, but not limited to, mannitol or sorbitol; salt-forming counterions such as, but not limited to, sodium; and/or nonionic surfactants such as, but not limited to, TWEEN.; polyethylene glycol (P
  • pharmaceutically acceptable carrier refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • a pharmaceutically acceptable carrier after administered to or upon a subject, does not cause undesirable physiological effects.
  • the carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and, preferably, capable of stabilizing it.
  • One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
  • a “subject” refers to a human and a non-human animal.
  • a non-human animal include all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals, such as birds, amphibians, reptiles, etc.
  • the subject is a human.
  • the subject is an experimental animal or animal suitable as a disease model (such as non-human primates).
  • a subject to be treated can be identified by standard diagnosing techniques for the disorder.
  • the present invention provides a method of reducing or preventing the establishment of a latent reservoir of HIV infected cells in a subject in need thereof (e.g., a subject infected with HIV or at risk of infection with HIV), thereby treating infection with a HIV infection, comprising administering to the subject a pharmaceutical composition comprising the HIV antibodies disclosed herein.
  • the compositions of the invention can include more than one antibody having the characteristics disclosed (for example, a plurality or pool of antibodies). It also can include other HIV neutralizing antibodies and/or active agent known in the art.
  • Subjects at risk for HIV-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to HIV in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of HIV-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • the patient is administered or provided a pharmaceutical formulation including an HIV antibody of the invention.
  • the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's latent viral reservoir).
  • the antibodies are administered to a human patient, in accord with known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibodies can be administered parenterally, when possible, at the target cell site, or intravenously.
  • antibody is administered by intravenous or subcutaneous administration.
  • Therapeutic compositions of the invention may be administered to a patient or subject systemically, parenterally, or locally. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • the antibodies may be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle.
  • a pharmaceutically acceptable, parenteral vehicle examples include, but are not limited, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers.
  • the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as, for example, buffers and preservatives.
  • the antibodies can be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
  • the dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection, for example, its therapeutic index, the patient, and the patient's history.
  • a therapeutically effective amount of an antibody is administered to a patient.
  • the amount of antibody administered is in the range of about 0.1 mg/kg to about 50 mg/kg of patient body weight.
  • about 0.1 mg/kg to about 50 mg/kg body weight (for example, about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • the progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • Other therapeutic regimens may be combined with the administration of the bNAb HIV antibody of the present invention.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • Such combined therapy can result in a synergistic therapeutic effect.
  • the parameters for assessing successful treatment and improvement in the disease are also readily measurable by routine procedures familiar to a physician.
  • treating or “treatment” or “alleviation” are used interchangeably and refer to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • it refers to administration of a compound or agent to a subject, who has a disorder (such as an HIV infection), with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
  • a disorder such as an HIV infection
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • the present invention relates to treatment of a subject in vivo using the above-described bispecific antibody such that growth and/or metastasis of cancerous tumors is inhibited.
  • the invention provides a method of inhibiting growth and/or restricting metastatic spread of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody.
  • Non-limiting examples of preferred cancers for treatment include chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, breast cancer, ovarian cancer, melanoma ⁇ e.g., metastatic malignant melanoma), renal cancer ⁇ e.g. clear cell carcinoma), prostate cancer ⁇ e.g. hormone refractory prostate adenocarcinoma), colon cancer and lung cancer ⁇ e.g. non-small cell lung cancer). Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention.
  • cancers examples include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma,
  • prevent refers to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • a “therapeutically effective amount” refers to the amount of an agent sufficient to effect beneficial or desired results.
  • a therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • Administration "in combination with” one or more further therapeutic agents include simultaneous (concurrent) and consecutive administration in any order.
  • compositions of this invention may be administered to humans and other animals by a variety of methods that may include continuous or intermittent administration. Examples of methods of administration may include, but are not limited to, oral, rectal, parenteral, intracisternal, intrasternal, intravaginal, intraperitoneal, topical, transdermal, buccal, or as an oral or nasal spray. Accordingly, the pharmaceutically effective compositions may also include pharmaceutically acceptable additives, carriers or excipients. Such pharmaceutical compositions may also include the active ingredients formulated together with one or more non-toxic, pharmaceutically acceptable carriers specially formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration according to standard methods known in the art.
  • parenteral administration refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intracisternal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • injectable mixtures are known in the art and comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate) and suitable mixtures thereof.
  • peptide refers to a peptide, polypeptide, or protein produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired peptide.
  • a “synthetic" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis.
  • fusion proteins containing one or more of the afore-mentioned sequences and a heterologous sequence.
  • a heterologous polypeptide, nucleic acid, or gene is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form. Two fused domains or sequences are heterologous to each other if they are not adjacent to each other in a naturally occurring protein or nucleic acid.
  • an "isolated" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated.
  • the polypeptide/protein can constitute at least 10% ⁇ i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • An isolated polypeptide/protein described in the invention can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods.
  • biological sample refers to a sample obtained from an organism (e.g., patient) or from components (e.g., cells) of an organism.
  • the sample may be of any biological tissue, cell(s) or fluid.
  • the sample may be a "clinical sample” which is a sample derived from a subject, such as a human patient.
  • samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • a biological sample may also be referred to as a "patient sample.”
  • a biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.
  • the term "contacting" and its variants when used in reference to any set of components, includes any process whereby the components to be contacted are mixed into same mixture (for example, are added into the same compartment or solution), and does not necessarily require actual physical contact between the recited components.
  • the recited components can be contacted in any order or any combination (or subcombination), and can include situations where one or some of the recited components are subsequently removed from the mixture, optionally prior to addition of other recited components.
  • contacting A with B and C includes any and all of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C.
  • Contacting a template with a reaction mixture includes any or all of the following situations: (i) the template is contacted with a first component of the reaction mixture to create a mixture; then other components of the reaction mixture are added in any order or combination to the mixture; and (ii) the reaction mixture is fully formed prior to mixture with the template.
  • non-cysteine residue refers to any natural or non-natural amino acid residue that is not cysteine and/or that does not form an SS-bond.
  • Examples include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arg
  • BiNAbs were generated using previously described approaches (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B.,
  • Antibodies, gpl40, and 2CC-core were generated by transient transfection of HEK293T or 293E cells, as previously described (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014)).
  • Antibodies were purified using PROTEF G SEPHAROSE 4 FAST FLOW (GE Healthcare); Strep-tagged and His-tagged proteins were purified using the STREP-TACTJN SUPERFLOW PLUS RESIN (Qiagen) and His- Tag isolation and pull-down dynabeads (ThermoFisher), respectively. Purified proteins were dialyzed in PBS and sterile filtered (0.22 ⁇ ). Purity was assessed by SDS-PAGE and Coomassie staining and was estimated to be >90%. Size exclusion chromatography (SEC) was performed using a SUPEROSE 6 INCREASE 10/300GL column (GE Healthcare) on an Akta Pure 25 HPLC system. Protein Tm was determined using the PROTEFN THERMAL SHIFT DYE Kit (ThermoFisher) following manufacturer's instructions on a QUANT STUDIO 12K FLEX real-time thermal cycler.
  • SEC Size exclusion chromatography
  • fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs by the IC50/80 titer of the biAb.
  • Predicted neutralization activity of a mix of two mAbs was determined by selecting the lowest IC50/80 titer of the two mAbs for a given virus.
  • gpl40- or 2CC-Core-coated (1 ⁇ g/ml) microtiter plates were used, as previously described (Bournazos, S. et al, Cell 158, 1243- 1253, doi: 10.1016/j .cell.2014.08.023 (2014)). Plate-bound IgG was detected by HRP- conjugated goat anti-human IgG (Fcy-specific, Jackson Immunoresearch), or goat anti- human kappa or lambda (Bethyl Laboratories).
  • IgG binding was detected using HRP-conjugated goat anti-human IgG (mouse IgG absorbed; Jackson Immunoresearch), as previously described (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014)).
  • HIV- 1 T251-1 was generated by cloning the T251-18 env gene (CRF02_AG; NIH AIDS Reagent program) to the NL4-3 HIV-1 vector backbone and produced by transfection in 293T cells, as previously described (Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)). HIV-1 virus preparations were quantified by p24 ELISA (Lenti-X p24 Rapid Titer Kit, Clontech), following manufacturer's recommendations.
  • mice were generated by intrahepatic human CD34 + HSCs injection of sublethally irradiated neonatal NRG mice, as previously described in Klein, F. et al, Nature 492, 118- 122, doi: 10.1038/naturel l604 (2012). Mice were screened at 6-8 wk of age for human leukocyte reconstitution by flow cytometry (as described in Bournazos, S.
  • mice with a measurable human CD45 + graft (10-15 weeks, males and females) were infected following i.p. injection of HIV-1 T251-18 (180 ng p24). Viral load was quantified 3-weeks post-infection and mice with viral loads >10 4 copies/ml were included in treatment experiments. Mice were randomly assigned to experimental groups and both groups had comparable baseline average viremia levels.
  • Antibodies (either 1 mg of 3BNC 117/PGT 135 biAb or 1 mg 1 : 1 mix of 3BNC117 (0.5 mg) and PGT135 (0.5mg)) were administered biweekly s.c. for 4 weeks.
  • Each experimental group consisted of 9 mice; a group size previously determined to sufficiently detect response to antibody therapy (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014) and Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012).
  • Plasma HIV-1 viral load was determined by quantitative reverse-transcriptase PCR as previously described in Bournazos, S.
  • Plasma-extracted viral RNA was reverse transcribed using the SUPERSCRIPT III first strand cDNA synthesis kit (Life Technologies) and the gpl20 sequence-specific primer 5'- T AGC A AT AGTTGTGTGGTC C-3 ' (SEQ ID NO: 37). Resulting cDNA was used as the template for PCR amplification using the following primer pairs: 5'-
  • Results from multiple experiments are presented as mean ⁇ standard error of the mean (SEM).
  • IC50 titers are presented as geometric mean ⁇ 95% CI.
  • Kruskal-Wallis test was used to test for differences in the IC50/80 titers, and where statistically significant effects were found, post hoc analysis using Dunn's multiple comparison test was performed.
  • Mann-Whitney non-parametric test two-sided was used. Data were analyzed with Graphpad Prism software (Graphpad) and P values of ⁇ 0.05 were considered to be statistically significant.
  • Pairing of the correct heavy chain with the respective light chain was achieved by swapping the CHI domain with the constant domain of the light chain (CL) for one mAb (10-1074), while preserving the wild-type domain organization for the other mAb (3BNC117) (Schaefer, W. et al, Proc Natl Acad Sci U S A 108, 11187-11192, doi: 10.1073/pnas.1019002108 (2011)).
  • This approach (CrossMab) had no measurable effect on the antigenic specificity and neutralization activity of the 10-1074 mAb (FIG. IB, FIG. 5 A, Table SI).
  • Heterodimerization of the heavy chain was achieved by introducing mutations in the CH3 domain that alter the physical and chemical properties of the two mAb heavy chains, favoring heterodimer formation (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B., Presta, L. G. & Carter, P., Protein Eng 9, 617-621 (1996)). Consistent with previous reports (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998) and Asokan, M. et al.
  • Table SI IC50 titers fag/ml) of 10-1074 hlgGl and 10-1074 CrossMab variant determined by TZMbl neutralization assay
  • Table S2 Comparison of the in vitro neutralization activity of human IgGl 3BNC117 and 10-1074 mAbs and 3BNC117/10-1074 biAb.
  • IC50 values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 titer ofthe most potent (lowest IC50) of the two parental mAbs (3BNC117 or 10-1074) by the IC50 titer ofthe biAb (3BNC117/10-1074).
  • the inventors extended their study by focusing on different regions of Env, including the Vl/2 epitope (targeted by the PG16 mAb), the V3 epitope (targeted by the 10-1074, PGT121 and PGT128 mAb), as well as antibodies against the gpl20/41 interface (PGT151 and 35022).
  • V1/2-V3 biNAbs PG16/10-1074, PG16/PGT121, PG16/PGT128) as well as the gpl20/41 interface biNAb, PGT151/35022 exhibited compromised neutralization potency and none of the combinations achieved the activity of their respective parental bNAbs (FIGs. 1D-G, Tables S3-S6).
  • Comparable findings have also been recently reported when the neutralization activity of biNAbs targeting similar epitopes on Env has been tested (Asokan, M. et al, J Virol 89, 12501-12512, doi: 10.1128/JVI.02097-15 (2015).).
  • Table S3 Comparison of the in vitro neutralization activity of human IgGl PG16 and PGT121 mAbs and PG16/PGT121 biAb.
  • IC50 values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) ofthe two parental mAbs (PG16 or PGT121) by the IC50 titer of the biAb (PG16/PGT121).
  • IC50 values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PG16 or PGT128) by the IC50 titer of the biAb (PG16 PGT128).
  • Table S5 Comparison of the in vitro neutralization activity of human IgGl PG16 and 10- 1074 mAbs and PG16/10-1074 biAb.
  • IC50 values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PG16 or 10-1074) by the IC50 titer of the biAb (PG16/10-1074).
  • IC50 values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PGT151 or 35022) by the IC50 titer of the biAb (PGT151/35022).
  • a key immune evasion mechanism of HIV-1 against host antibody responses is the remarkably low density of Env molecules on the viral surface, as well as the unique Env trimeric architecture, which preclude high-avidity bivalent interactions of IgG (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010)). It is therefore likely that the tested biNAbs would exhibit predominantly monovalent binding to their respective epitopes, possibly accounting for the lack of additive, synergistic activity. Indeed, given the relative rigidity and the short length of the hinge domain of IgGl, concurrent binding of the two Fab arms within the same Env trimer is largely restricted.
  • IgG3 encompasses an exceptionally long and flexible hinge domain, with distinct structural and functional characteristics (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997) and Roux, K. H., et al, J Immunol 761, 4083-4090 (1998)) (FIG. 2A). It is comprised of a 17mer amino acid sequence followed by three 15-mer repeats that are highly homologous to the IgGl hinge structure and represent genomic duplication events of the ancestral hinge-encoding exon, conserved among all other IgG subclasses (Roux, K.
  • the inventors aimed to develop biNAbs with increased Fab domain flexibility, using the IgG3 hinge as template.
  • IgG3C- an "open" IgG3-based hinge variant
  • the inventors generated an "open" IgG3-based hinge variant (IgG3C-), in which all the cysteine residues have been replaced with serines with the exception of the last two, CH2 proximal residues that are used to maintain the structural integrity of the Fc domain through inter-heavy chain disulfide bonding (FIG. 2A).
  • Hinge domain variants of 10-1074 IgGl were expressed as chimeric molecules, in which the wild-type hinge domain (IgGl) was replaced with that of either IgG3 or the IgG3C- variant. With the exception of the hinge domain, all other domains of the constant region (CHI, CH2, CH3) were of the IgGl subclass to preserve the effector function and half-life of wild-type IgGl . Indeed, hinge domain variants of 10- 1074 demonstrated comparable binding affinity to the different classes of human and mouse FcyRs, suggesting a minimal role for the hinge region in Fc-FcyR interactions (Extended Data Table 1). Likewise, no differences among the hinge domain variants were noted in terms of protein stability and in vivo pharmacokinetics (FIG. 2B and FIG. 6).
  • Values represent KD ⁇ M) determined by surface plasmon resonance using soluble human or mouse FcyR ectodomains.
  • IgG3C- hinge variant of the 3BNC117/10-1074 biNAb presented substantially improved activity.
  • IgG3C- hinge variant of the 3BNC117/10-1074 biNAb presented substantially improved activity.
  • IC50 ⁇ g/ml 0.242 vs. 0.110
  • ICso ⁇ g/ml 0.717 vs. 0.388 for IgGl vs.
  • IgG3C-biNAb 15 IgG3C-, respectively.
  • the observed neutralization breadth and potency of the IgG3C-biNAb was comparable to that expected from the mix of the two parental antibodies (FIG. 2C, Tables S7-S8).
  • IgGl biNAb exhibited the average neutralization potency of the two parental bNAbs (3BNC117 and 10-1074)
  • IgG3C- biNAb demonstrated activity equal or in some cases better than that of the parental
  • IC50 and ICso values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 or ICso titer of the IgG3C- with that of IgGl hinge variant biAb.
  • Table S8 Comparison of the in vitro neutralization activity of IgG3C5 hinge variants of 3BNC117and 10-1074 mAbs and 3BNC117/10-1074 biAb.
  • In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 and ICso values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs (3BNC117 or 10-1074) by the ICso / so titer of the biAb (3BNC117/10-1074).
  • IC50 values are expressed as ⁇ g/ml.
  • bNAbs were cloned as IgG3C- hinge domain variants and biNAbs were generated with non- overlapping epitope specificities. No difference in the neutralization activity was observed between IgGl and IgG3C- hinge variants for all the parental, monospecific antibodies (FIG. 7, Extended Data Table 2 below, and Table S9 above).
  • biNAb combinations that showed evidence for synergistic activity were selected for further analysis, based on their epitope specificities, evidence for efficient expression, sufficient in vivo half-life, and long-term protein stability. These combinations included: PGT151/10-1074, 8ANC195/PGT128, and 3BNC117/PGT135 biNAbs. Evaluation of their neutralization activity against an extended multiclade virus panel revealed that the 8ANC195/PGT128 biNAb combination presented modest synergistic activity, while no differences were noted for the PGT151/10-1074 biNAb (FIGs. 4A-B, Tables S11-S12 below).
  • 3BNC1 17/PGT135 biNAb exhibited augmented neutralization breadth and potency, surpassing the activity of the parental bNAbs (3BNC117 and PGT135) (FIG. 4C, Extended Data Table 3).
  • 3BNC 117/PGT 135 biNAb demonstrated lower IC50 and IC80 titers compared to the most potent of the parental bNAbs (3BNC117 or PGT135) for each tested virus strain.
  • the 3BNC117/PGT135 IgG3C- biNAb was capable of neutralizing over 93% of the tested viruses, with an average (geometric mean) IC50 of 0.036 ⁇ g/ml, representing one of the most potent anti-HIV-1 Env antibodies characterized so far.
  • Extended Data Table 2 In vitro neutralization activity of hinge variants of anti-HIV-1 Env mAbs against a multiclade virus panel.
  • In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 values are expressed as ⁇ .
  • Table S10 In vitro neutralization activity oflgGSC- hinge variants of mAbs and biAbs against a multiclade virus panel.
  • In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 and ICso values are expressed as ⁇ .
  • Table Sll Comparison of the in vitro neutralization activity oflgGSC- hinge variants of 8ANC195 andPGT128 mAbs and 8ANC195/PGT128 biAb
  • IC50 and ICso values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs (8ANC195 or PGT128) by the ICso/so titer of the biAb (8ANC195/PGT128).
  • Table SI 2 Comparison of the in vitro neutralization activity of IgGSCl hinge variants of PGT151 and 10-1074 mAbs and PGT151/10-1074 biAb.
  • IC50 and ICso values are expressed as ⁇ .
  • Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest ICso/so) of the two parental mAbs (PGT151 or 10-1074) by the IC 5 o/so titer of the biAb (PGT151/10-1074).
  • In vitro neutralization activity was determined by standardized TZM ⁇ bl assay. IC50 and ICso values are expressed as ⁇ g/ml. Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest ICso / so) of the two parental mAbs (3BNC117 or PGT135) by the ICso / so titer of the biAb (3BNC117/PGT135). ND: not determine.
  • a tier 2 HIV-1 virus stain (T251-18) was selected that was highly sensitive (IC50: ⁇ 0.023 to the 3BNC117/PGT135 biNAb, while exhibiting modest sensitivity or resistance for 3BNC117 (ICso: 0.219 and PGT135 (ICso: >50 ⁇ ) respectively.
  • HIV-l T251"18 -infected humanized mice were treated either with a mix (1 : 1) of 3BNC117 and PGT135 bNAbs or with 3BNC 117/PGT 135 biNAb (all in IgG3C- hinge variant format).
  • FIG. 9A- B Quantitation of plasma viremia revealed that 3BNC117/PGT135 biNAb treatment decreased viremia by an average of 1.5 logw during the treatment period (FIGs. 4E-G) with the majority (7/9) of treated animals exhibiting substantially reduced plasma viremia levels ( ⁇ logw viremia ⁇ -1.0).
  • the synergistic activity observed for the 3BNC 117/PGT 135 IgG3C- biNAb might actually reflect the capacity of this biNAb combination for bivalent binding of the two Fab arms accomplished by the flexible IgG3C- hinge structure.
  • comparison of the gpl20-bound PGT135 Fab crystal structure to other anti-V3 bNAbs (PGT122, or PGT128) revealed a unique orientation and angle for PGT135 (Kong, L. et al. 2013, Nat Struct Mol Biol 20, 796-803) that when combined with the 3BNC117 bNAb, as in the case of 3BNC117/PGT135 IgG3C- biNAb, would facilitate intra-trimeric bivalent interactions (FIGs.
  • 3BNC117/PGT135 biNAbs and their respective parental bNAbs were expressed as variants encompassing the hinge domain structure of wild- type IgGl, IgG3, or IgG3C- (open hinge structure based on wild-type IgG3 sequence) and their neutralization activity was assessed against a multiclade, 20-strain panel.
  • the activity of a 1 : 1 mix of the two parental bNAbs was compared among the different hinge domain variants, no significant differences were noted, with all the hinge variants exhibiting comparable neutralization activity (FIGs. 10A and 10B).
  • 3BNC117/PGT135 biNAb demonstrated significantly augmented activity only in the IgG3C-, but not in the IgGl or IgG3 hinge domain format, indicating that the synergistic activity of the 3BNC117/PGT135 biNAb is dependent upon the unique structure and flexibility of the IgG3C- hinge variant.
  • the antibody ti/ 2 for 3BNC117/PGT135 IgG3C- biNAb was calculated to range from 86.37 - 87.46 h (3.5-3.6 d), which is comparable to that of anti-HIV Env IgGl monospecific bNAbs (previously determined in Shingai et al, J. Exp. Med. 2014 Vol. 211 No. 10 2061-2074). See FIG. 12.

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Abstract

La présente invention concerne des anticorps bispécifiques et, en particulier, des anticorps largement neutralisants anti-VIH et leurs utilisations.
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